Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria

Figure 6

Overexpression of Bcl-2 attenuates IFNγ- and TNFα-induced cytotoxicity in MC3T3-E1 cells. (a) Western blot for Bcl-2 in cells stably expressing Bcl-2. Total cell lysates were prepared from cells stably transfected with control vector (pcDNA3) or a Bcl-2 expression vector (pCMV-Bcl-2), of which 20 μg protein was subjected to 12% SDS-PAGE and analyzed with antibodies to Bcl-2. The blot was stripped and reprobed with anti-tubulin to confirm loading of equal amounts of total protein. (b) Effect of IFNγ and TNFα costimulation on the viability of cells stably expressing Bcl-2. Cells stably transfected with control vector or pCMV-Bcl-2 were seeded in 96-well plates, incubated for 5 days to form a confluent monolayer, and costimulated with 10 ng/ml IFNγ and 5 ng/ml TNFα. Cell viability was monitored over time. Data represent the mean ± SEM of three independent experiments. versus control cells by Student’s -test.

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